Two pyrenylalanines in dihydrofolate reductase form an excimer enabling the study of protein dynamics

Shengxi Chen; Lin Wang; Nour Eddine Fahmi; Stephen J. Benkovic; Sidney Hecht

doi:10.1021/ja307179q

Two pyrenylalanines in dihydrofolate reductase form an excimer enabling the study of protein dynamics

Shengxi Chen, Lin Wang, Nour Eddine Fahmi, Stephen J. Benkovic, Sidney Hecht

Research output: Contribution to journal › Article › peer-review

30 Scopus citations

Abstract

Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl- tRNA_CUA. Excimer formation (λ_ex 342 nm; λ_em 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.

Original language	English (US)
Pages (from-to)	18883-18885
Number of pages	3
Journal	Journal of the American Chemical Society
Volume	134
Issue number	46
DOIs	https://doi.org/10.1021/ja307179q
State	Published - Nov 21 2012

ASJC Scopus subject areas

Catalysis
General Chemistry
Biochemistry
Colloid and Surface Chemistry

Access to Document

10.1021/ja307179q

Cite this

@article{24de7041a2d94ff1a0b132cd06295562,

title = "Two pyrenylalanines in dihydrofolate reductase form an excimer enabling the study of protein dynamics",

abstract = "Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl- tRNACUA. Excimer formation (λex 342 nm; λem 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.",

author = "Shengxi Chen and Lin Wang and Fahmi, {Nour Eddine} and Benkovic, {Stephen J.} and Sidney Hecht",

year = "2012",

month = nov,

day = "21",

doi = "10.1021/ja307179q",

language = "English (US)",

volume = "134",

pages = "18883--18885",

journal = "Journal of the American Chemical Society",

issn = "0002-7863",

publisher = "American Chemical Society",

number = "46",

}

TY - JOUR

T1 - Two pyrenylalanines in dihydrofolate reductase form an excimer enabling the study of protein dynamics

AU - Chen, Shengxi

AU - Wang, Lin

AU - Fahmi, Nour Eddine

AU - Benkovic, Stephen J.

AU - Hecht, Sidney

PY - 2012/11/21

Y1 - 2012/11/21

N2 - Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl- tRNACUA. Excimer formation (λex 342 nm; λem 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.

AB - Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl- tRNACUA. Excimer formation (λex 342 nm; λem 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.

UR - http://www.scopus.com/inward/record.url?scp=84869384366&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84869384366&partnerID=8YFLogxK

U2 - 10.1021/ja307179q

DO - 10.1021/ja307179q

M3 - Article

C2 - 23116258

AN - SCOPUS:84869384366

SN - 0002-7863

VL - 134

SP - 18883

EP - 18885

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 46

ER -

Two pyrenylalanines in dihydrofolate reductase form an excimer enabling the study of protein dynamics

Abstract

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this